1. Field of the Invention
The present invention relates generally to medicine. More specifically, the invention is directed to methods relating to treating or preventing dementia.
2. Background Information
Dementia is a neurological disease that results in loss of intellectual capacity and is associated with widespread reduction in the number of nerve cells and brain tissue shrinkage. Memory is the mental capacity most often affected. The memory loss may first manifest itself in simple absentmindedness, a tendency to forget or misplace things, or to repeat oneself in conversation. As the dementia progresses, the loss of memory broadens in scope until the patient can no longer remember basic social and survival skills and function independently. Dementia can also result in a decline in the patient's language skills, spatial or temporal orientation, judgment, or other cognitive capacities. Dementia tends to run an insidious and progressive course.
Dementia results from a wide variety of distinctive pathological processes. The most common pathological process to cause dementia is Alzheimer's disease, which results in Alzheimer's-type dementia (AD). The second most common cause is multi-infarct, or vascular dementia, which results from hypertension or other vascular conditions. Dementia can also result from infectious disease, such as in Creutzfeldt-Jakob disease. Dementia occurs in Huntington's disease, which is caused by an autosomal dominant gene mutation, and in Parkinson's disease, which is associated with a motor disorder. Dementia also occurs from head injury and tumors.
Rare before age 50, AD affects nearly half of all people past the age of 85, which is the most rapidly growing portion of the United States population. As such, the current 4 million AD patients in the United States are expected to increase to about 14 million by the middle of the next century.
No method of preventing AD is known and treatment is primarily supportive, such as that provided by a family member in attendance. Stimulated memory exercises on a regular basis have been shown to slow, but not stop, memory loss. A few drugs, such as tacrine, result in a modest temporary improvement of cognition but do not stop the progression of dementia.
A hallmark of AD is the accumulation in brain of extracellular insoluble deposits called amyloid plaques, and abnormal lesions within neuronal cells called neurofibrillary tangles. The presence of amyloid plaques, together with neurofibrillary tangles, are the basis for definitive pathological diagnosis of AD. Increased plaque formation is associated with increased risk of AD.
The major components of amyloid plaques are the amyloid .beta.-peptides, also called A.beta. peptides, which consist of three proteins having 40, 42 or 43 amino acids, designated as the A.beta..sub.1-40, A.beta.-.sub.1-42, and A.beta..sub.1-43 peptides. The amino acid sequences of the A.beta. peptides are known and the sequence of the A.beta..sub.1-42 is identical to that of the A.beta..sub.1-40 peptide, except that the A.beta..sub.1-42 peptide contains two additional amino acids at its carboxyl (COOH) terminus. Similarly, the amino acid sequence of the A.beta..sub.1-43 peptide is identical to that of the A.beta..sub.1-42 peptide except that the A.beta..sub.1-43 peptide contains one additional amino acid at its carboxyl terminus. The A.beta. peptides are thought to cause the nerve cell destruction in AD, in part, because they are toxic to neurons in vitro and in vivo.
The A.beta. peptides are derived from larger amyloid precursor proteins (APP proteins), which consist of four proteins, designated as the APP.sub.695, APP.sub.714, APP.sub.751, and APP.sub.771 proteins, which contain 695, 714, 751 or 771 amino acids, respectively. The different APP proteins result from alternative ribonucleic acid splicing of a single APP gene product. The amino acid sequences of the APP proteins are also known and each APP protein contains the amino acid sequences of the A.beta. peptides.
Proteases are believed to produce the A.beta. peptides by recognizing and cleaving specific amino acid sequences within the APP proteins at or near the ends of the A.beta. peptides. Such sequence specific proteases are thought to exist because they are necessary to produce from the APP proteins the A.beta..sub.1-40, A.beta..sub.1-42, and A.beta.1-43 peptides consistently found in plaques.
But the proteases have not been isolated. Nonetheless, they have been named "secretases" because the A.beta. peptides which they produce are secreted by cells into the extracellular environment. Moreover, the secretases have been named according to the cleavages that must occur to produce the A.beta. peptides. The secretase that cleaves the amino terminal end of the A.beta. peptides is called the .beta.-secretase and that which cleaves the carboxyl terminal end of the A.beta. peptides is called the .gamma.-secretase. The .gamma.-secretase determines whether the A.beta..sub.1-40, A.beta..sub.1-42, or A.beta..sub.1-43 peptide is produced (see FIG. 1). But since the secretases have not been isolated, the terms .beta.-secretase and .gamma.-secretase each could relate to one or several protease species.
In addition to the A.beta. peptides, proteolytic cleavage of another specific amino acid sequence within the APP proteins is known to occur and to produce .alpha.-APP and 10 kilodalton (kDa) fragments. That amino acid sequence lies within the A.beta. peptide amino acid sequence of the APP proteins. Like the .beta.-secretase and the .gamma.-secretase, the protease responsible for that cleavage has also not been isolated but has been named the .alpha.-secretase (see FIG. 1). Significantly, the products produced by the .alpha.-secretase cleavage, the .alpha.-APP and the 10 kilodalton (kDa) fragments, do not form senile plaques.
Proteases can be isolated from tissue homogenates or lysed cell samples, but those samples can contain the proteases from cell organelles in which the product is not produced, but which may be able to cleave in vitro the precursor protein to produce the product. Thus, a problem in using such samples to isolate the secretases has been that proteases which produce the A.beta. peptide in vitro, but not in vivo, may be erroneously isolated.
The problem can be avoided by isolating the secretase from cell organelles in which the APP proteins are processed in vivo. A cell organelle thought to be a site in which such processing occurs is the secretory vesicles of brain neuronal cells. But methods have not been developed to obtain sufficient amounts of pure secretory vesicles from neuronal cells to assay for secretase activity in those vesicles.
Large amounts of pure secretory vesicles can be obtained from chromaffin cells, neuroendocrine cells of the adrenal medulla, and have been used to obtain proteases. For example, carboxypeptidase H (CPH), prohormone thiol protease (PTP), and the prohormone convertases (PC1 and PC2), which process precursor proteins into peptides having opiate activity have been obtained from such vesicles. But chromaffin cells have not been shown to produce the A.beta. peptides or have secretase activity.
The .beta.-secretase, .gamma.-secretase, and .alpha.-secretase must be isolated to understand how the neurotoxic A.beta. peptides are produced so that AD can be prevented or treated. To isolate the secretase, new methods are needed for assaying the proteolytic activity of secretases in substantially purified preparations of the cell organelles in which the APP protein is processed in vivo. Moreover, new screening methods for selecting agents that affect the proteolytic activity of the secretases are needed to develop new pharmaceuticals for treating or preventing AD. Further, such new methods need to be applied and the secretases isolated.
The invention satisfies these needs by providing new methods of determining the proteolytic activity of secretases and isolating secretases having that activity. The invention also provides new screening methods for selecting agents that affect the activity of such secretases. Moreover, the invention discloses novel .beta.-secretases obtained by such methods as well as methods of selecting agents inhibiting production of A.beta. peptides by inhibiting the activity of those .beta.-secretases.